Project Summary Gene fusions of the orphan receptor tyrosine kinase, ROS1, are expressed in a range of malignancies. My lab and other labs have demonstrated that ROS1 gene fusions drive neoplastic transformation of cells in vitro and are tumorigenic in vivo when concurrent with loss of tumor suppressor (TP53). It is thought that these gene fusions transform cells via uncontrolled ROS1 kinase activity. Importantly, clinical implementation of the first- generation ALK and ROS1 tyrosine kinase inhibitor (TKI), crizotinib, has resulted in significant reduction of tumor burden in patients with ROS1-fusion positive tumors. However, the efficacy of crizotinib treatment is often short-lived as resistance to the drug emerges after a period of time. Therefore, ongoing research efforts will be required to discover second-line or combination therapeutic options. Clinical resistance to crizotinib treatment has taken on two forms: 1) aberrations in alternate signaling pathways that compensate for any loss in ROS1 kinase signaling (?bypass pathway aberrations?) and 2) loss of TKI potency due to point mutations in the ROS1 kinase domain. While both forms of resistance are equivalent in terms of incidence, little attention has been provided towards understanding bypass pathway aberrations. Notably, a patient that our collaborators at Memorial Sloan Kettering Cancer Center (MSKCC) are treating has stopped responding to crizotinib therapy for ROS1-fusion positive non-small-cell lung cancer after initially seeing tumor regression. Sequencing of the crizotinib-resistant tumor found a copy-number amplification (CNA) in the transcription factor, MYC, that was absent in the initial tumor. I hypothesize that CNA of MYC represents a bypass pathway aberration that has reduced the efficacy of crizotinib treatment in this patient and that this phenomenon can occur in other tumors. To study this, I will 1) reversibly perturb MYC in cells derived from the patient tumor to determine if ROS1-TKI sensitivity is restored and 2) overexpress MYC in a crizotinib- sensitive, ROS1-fusion expressing human broncho-epithelial cell line to observe if ROS1-TKI resistance appears. Unlike bypass pathway aberrations, much research has been conducted to characterize ALK and ROS1 kinase point mutations that confer resistance to crizotinib. This has aided the design of second-generation ALK/ROS1- TKIs like lorlatinib that can bind to the ROS1 kinase domain even in the presence of these mutations. However, I hypothesize that resistant point mutations will develop towards the new TKIs and that predicting them will inform future drug design. To study this, I will perform a forward mutagenesis screen in a lorlatinib-sensitive ROS1-fusion cell line to identify mutations in ROS1 that confer resistance to lorlatinib. Taken together, achieving the objectives of this project will: 1) discover a bypass pathway aberration that causes resistance to the ALK/ROS1-TKI, crizotinib, and 2) identify ROS1 point mutations that confer resistance to a next-generation ROS1-TKI, lorlatinib. This will help combat future ROS1-TKI resistance in patients.